In the United States, more than 12 million new cases of sexually transmitted diseases (STDs) occur each year. Of the top 10 reportable diseases in the United States, five are STDs including chlamydia, gonorrhea, syphilis, the Acquired Immune Deficiency Syndrome (AIDS) and hepatitis B virus (HBV) infection. Human immunodeficiency virus (HIV) infection is a chronic disease that erodes the immune system, ultimately resulting in AIDS and death. There is currently no cure for AIDS and many cases of HBV resist treatment.
In the case of AIDS, the World Health Organization recently estimated there are 85 million people worldwide infected with the human immunodeficiency virus (HIV), the virus that causes AIDS. Hepatitis B virus infections affect 5 times more people than HIV. It is estimated that 350 million individuals are chronically infected with HBV and that 1 to 2% will die each year from complications associated with infection, with the majority of these deaths occurring from cirrhosis of the liver and hepatocellular carcinoma.
Replication of HIV is measured by plasma RNA viral load, and in untreated patients, it is estimated that 10 billion virions are produced daily (Levin et al., Science 1997, 275(5298):334-43). If untreated, the infection damages the immune system, resulting in a decline in the CD4 count and subsequent development of opportunistic infections or AIDS related malignancies. Prophylactic regimens can be used to minimize the morbidity and mortality associated with opportunistic infections, such as pneumocystis carinii or mycobacterium avium. 
HIV-infected individuals can be shown to have a variety of immunologic responses to HIV, including cytotoxic T cell responses (to a variety of viral products, including reverse transcriptase), antibody responses, and antibody-dependent cellular cytotoxicity. However, even with these responses, disease progression usually occurs, and patients progress to full-blown AIDS and death.
Vaccines are among the most effective strategies for preventing and controlling viral infections. Vaccines have been proven effective primarily against viruses causing acute, self-limited infections. For these it has been sufficient for the vaccine to mimic the natural virus, such as a live, attenuated virus. However, generally, in chronic viral infections, such as HIV, HBV, Hepatitis C virus (HCV) or human herpesvirus infection, the virus does not elicit an immune response sufficient to eradicate the infection (Berzofsky et al., Nat Rev Immunol 2001, 1(3):209-19; Berzofsky et al., J Clin Invest 2004, 114(4):450-62). Therefore, a vaccine that just mimics the natural infection is not likely to be adequate to induce protection. Also, there is much concern about the use of live attenuated viruses for vaccination against these diseases. Although advances in molecular biology have raised great hope for the development of new vaccine strategies and much effort has been invested in this endeavor, recombinant viral protein vaccines, such as a hepatitis B surface antigen vaccine, have been a rarity (Hilleman, Vaccine 2001, 19:1837-1848).
In the last 5-10 years, however, many new vaccine strategies based on improved ways of inducing antibodies or inducing cytotoxic T lymphocytes (CTLs) have been designed. CTLs detect and destroy cells infected with virus and thereby control and ultimately clear infection. CTLs can detect any viral protein made within an infected host—even when this viral protein is not present on the cell surface. CTLs are also able to respond to peptide fragments of these viral proteins produced by proteasomal cleavage and transported to the endoplasmic reticulum. Here, these peptides bind to newly synthesized class I MHC proteins, such as HLA-A, -B, and -C in humans, which carry the peptides to the cell surface and present them to T-cells (Berzofsky et al., J Clin Invest 2004, 114(4):450-62).
Typically, viral antigens are presented by MHC class I molecules in the form of 8-9 amino acid epitopes that act to stimulate a CTL response. The major CTL immune response to HIV is spread over the gene products Env, Gag, Nef, Vif, Tat, and Pol (Hadida et al., J Immunol 1995, 154(8):4174-86); Walker et al., Science 1988, 240(4848):64-6; Plata et al., Nature 1987, 328(6128):348-51; Koenig et al., Proc Natl Acad Sci USA 1988, 85(22):8638-42; Lamhamedi-Cherradi et al., Aids 1992, 6(11):1249-58).
In protection against HIV, CD8+ CTL play a major role. Many HIV-infected long-term non-progressors have expressed a high level of HIV-specific CTLs. The most direct evidence that CD8+ T lymphocytes, especially CTLs, are involved in controlling HIV infection comes from studies of HIV-infected chimpanzees. Here, depletion of CD8+ T cells in vivo led to an increase in viral load that was later reversed when T cells reappeared (Castro et al., Clin Immunol Immunopathol 1992, 65:227-233). Similar observations were made for SIV in macaques (Schmitz et al., Science 1999, 283:857-860; Jin et al., J Exp Med 1999, 189:991-998).
Although virus-specific CTLs can be elicited by peptides, one approach is to induce endogenous expression of a viral antigen in an antigen-presenting cell, such as a dendritic cell. This seems to be an efficient way to load class I MHC molecules with peptides for presentation to CD8+ T cells (Berzofsky et al., J Clin Invest 2004, 113:1515-1525; Berzofsky et al., J Clin Invest 2004, 114(4):450-62).
However, mutations of HIV allowing the virus to escape from immune control mediated by CTLs are a major concern. This has led to the consideration of new vaccine strategies (reviewed in Berzofsky et al., Nat Rev Immunol 2001, 1(3):209-19). Viral sequences evolving under immune selective pressure would not likely have optimal HLA molecule-binding epitopes. Thus, viral proteins are not naturally selected for high affinity to MHC binding sequences. Indeed, if there is any selection, it is likely to be negative in nature, allowing the virus to escape. Thus, one effective strategy toward development of new generation vaccines is to modify viral epitope sequences to improve the CTL response.
One such strategy involved the creation by sequence modification of enhanced epitopes that bind with higher affinity to MHC molecules. As a possible solution for eliciting an immune response against HIV, Okazaki et al. (J Immunol 2003, 171(5):2548-55) used an epitope-enhancement strategy involving a conserved CTL epitope in HIV reverse transcriptase (RT), VIYQYMDDL (RT-WT, amino acid residues 179-187; SEQ ID NO:1) for the induction of antiviral protection in HLA-A2 transgenic mice mediated by human HLA-A2-restricted CTLs. This strategy involved modifying the conserved epitope sequence to improve binding to human leukocyte antigen (HLA) molecules, such as HLA-A2, which is the most common human class I MHC molecule (Okazaki et al., J Immunol 2003, 171(5):2548-55). Specifically, Okazaki et al., designed two epitope-enhanced peptides based on affinity for HLA-A2, one substituted in anchor residues (RT-2L9V) and the other also with tyrosine at position 1 (RT-1Y2L9V) and examined the balance between HLA binding and T cell recognition. This study demonstrated that the enhanced CTL epitope, in which the anchor residues were modified for enhanced binding to the HLA-A2 molecule, can induce CTL more efficiently while maintaining full crossreactivity to the original viral epitope.
We have previously succeeded in improving the affinity of a hepatitis C core epitope for HLA-A2.1 (Sarobe et al., J. Clin Invest 1998, 102:1239-1248) and of a helper epitope for murine class II MHC (Ahlers et al., Proc Natl Acad Sci USA 1997, 94:10856-10861; Ahlers et al., J Clin Invest 2001, 108:1677-1685). Further, an epitope-enhanced melanoma peptide has shown efficacy in human clinical trials (Rosenberg et al., Nat Med 1998, 4:321-327).
In the case of HIV antiviral therapy has been utilized successfully to control viral replication. Although mortality rates from AIDS are dropping due to new drug therapies, AIDS remains the second leading cause of death in adults between the ages of 29 and 40. Combination anti-HIV therapy is now the standard of care for people infected with HIV and has dramatically decreased the number of AIDS-related deaths. There are 12 anti-HIV drugs available by prescription. These anti-HIV drugs fall into three categories: (i) nucleosides analogs, which include zidovudine, didanosine, zalcitabine, stavudine and lamivudine (or 3TC); (ii) protease inhibitors, which include indinavir, nelfinavir, saquinavir, ritonavir and amprenavir (Akhteruzzaman et al., Antiviral Res 1998, 39:1-23) and (iii) non-nucleoside reverse transcriptase inhibitors, which include nevirapine, delavirdine and efavirenz.
Compared to HIV, there are presently only two licensed therapies for chronic hepatitis B virus infection, interferon and lamivudine. Lamivudine is part of many antiretroviral regimens due to its favorable pharmokinetics, low toxicity, and high potency against HIV. Other drugs are currently under clinical trials including famciclovir, lobucavir and adefovir. However, many studies have shown that most patients relapse after completion of therapy and develop resistance to the drugs.
However, a major barrier to the anti-viral drug treatment of HIV infections is that the high degree of genetic variation and high levels of viral replication often lead to the emergence of drug-resistant variants during treatment. Drug resistance is a major concern in the treatment of HIV and Hepatitis B virus infections. Once a mutation conferring drug resistance occurs, the virus grows unchecked to become the dominant strain of the virus in the affected individual, and the drug becomes progressively less effective against the new strain. In clinical studies, resistance to 3TC was observed in nearly all patients who received 3TC monotherapy for more than 12 weeks (Schuurman et al., J Infect Dis 1995, 171:1411-1419).
A common target for HIV therapy is the reverse transcriptase (RT) of HIV. However, mutations of HIV leading to escape from RT inhibitors and other anti-HIV drugs have been observed. An important component of triple drug anti-AIDS therapy is the (−) enantiomer of 2′,3′-dideoxy-3′-thiacytidine (3TC, lamivudine). High-grade resistance to this nucleoside RT inhibitor is initially associated with the appearance of a resistant virus variant containing an M184I alteration in the RT sequence, i.e., a substitution of methionine to isoleucine at position 184 of the HIV RT. This transiently appearing variant is then rapidly replaced by an HIV variant carrying an M184V substitution, i.e., an amino acid substitution of methionine to valine at position 184 of the RT (Boucher et al., Antimicrob Agents Chemother 1993, 37(10):2231-2234; Gao et al., Antimicrob Agents Chemother 1993, 37(6):1390-1392; Sarafianos et al., Proc Natl Acad Sci USA 1999, 96(18):10027-10032; Schuurman et al., J Infect Dis 1995, 171(6):1411-1419; Wainberg et al., AIDS 1995, 9(4):351-357; Johnson et al., Top HIV Med 2003, 11(6):215-221). These mutations result in a >1,000 fold decrease in lamivudine sensitivity (Kanagawa et al., Science 1993, 262:240-2).
Amino residue 184 is contained within the catalytic site of RT, a highly conserved motif of YMDD (SEQ ID NO:2). These lamivudine escape mutations are located within an HLA-A2-restricted CTL epitope, VIYQYMDDL (RT-WT; SEQ ID NO:1) defined in a long-term non-progressing HIV-1 infected individual (Harrer et al., J Infect Dis 1996, 173:476-479). The selection of high level resistance to lamivudine can occur within weeks in patients with incomplete HIV suppression. In addition, the M184I and M184V mutations are also associated with reduced sensitivity to didanosine, zalcitabine and abacavir.
HIV viruses with mutations at residue 184 of RT generally arise only under drug pressure. Because these mutations adversely affect the function of RT and the replicative capacity of HIV-1, these mutations are infrequently found in wild-type virus (Back et al., EMBO J 1996, 15:4040-4049; Wainberg et al., Science 1996, 271:1282-1285).
Sarafianos et al. (Proc Natl Acad Sci USA 1999, 96(18):10027-32) determined the crystal structure of a 3TC-resistant mutant HIV-1 RT (M184I) and concluded that a steric conflict between the oxathiolane ring of the nucleotide analog 3TCTP and the side chain of beta-branched amino acids (Val, Ile, Thr) at position 184 perturbs inhibitor binding, leading to a reduction in incorporation of the analog. Their model can also explain the 3TC resistance of analogous polymerase mutants. For example, this model suggests that, like HIV-1 RT, a mutation of the methionine of the YMDD (SEQ ID NO:2) motif of hepatitis B polymerase (M552 in HBV) to a beta-branched amino acid would cause a steric conflict with the oxathiolane ring of 3TC. However, additional mutations outside the YMDD (SEQ ID NO:2) motif of the HBV polymerase have been reported to confer 3TC resistance. This suggests that other interactions may also affect HBV polymerase sensitivity to 3TCTP (Chang et al., J Biol Chem 1992, 267(2):13938-13942; Fu and Cheng, Biochem Pharmacol 1998, 55(10):1567-1572). For example, Lindstrom et al. (J Clin Microbiol 2004, 42(10):4788-4795) reported lamivudine-resistant HBV mutants that display specific mutations in the YMDD (SEQ ID NO:2) motif of the viral polymerase, such as methionine 204 to isoleucine or valine. They found that the latter mutation is often accompanied by a compensatory leucine-to-methionine change at codon 180.
Similar to the HIV-1 and HBV enzymes, simian immunodeficiency virus RT develops resistance to 3TC through a methionine to isoleucine or methionine to valine mutation in the YMDD (SEQ ID NO:2) motif (Cherry et al., Antimicrob Agents Chemother 1997, 41(12):2763-2765). None of the 3TC-resistant clones displayed resistance to 3′-azido-3′-deoxythynidine (AZT) or to the protease inhibitors indinavir and saquinavir, suggesting that resistance to these drugs involves other amino acid residues within RT or protease respectively.
Further, a methionine to threonine mutation in the YMDD (SEQ ID NO:2) motif of feline immunodeficiency virus (FIV) RT confers resistance to oxathiolane nucleosides (Smith et al., J Virol 1997, 71(3):2357-2362). Another mutation, a proline to serine mutation at position 156 of FIV RT was resistant to 3TC, AZT, and the combination of 3TC and AZT (Smith et al., J Virol 1998, 72(3):2335-40).
Schmitt et al. (AIDS 2000, 14(6):653-658) tested whether the M184V mutation of HIV RT represented a new CTL epitope and studied recognition of this epitope in 28 HLA-A2-positive HIV-1-infected patients. In one 43-year-old HIV-infected patient they could isolate a CTL line recognizing the peptide VIYQYVDDL (RT-M184V; SEQ ID NO:3) in conjunction with HLA-A2. The CTL clone also recognized the RT-M184I mutation, but failed to recognize the wild-type epitope, VIYQYMDDL (RT-WT; SEQ ID NO:1). Schmitt et al. concluded that CTL can specifically recognize lamivudine-resistant HIV-1 variants and that the cellular response could have an important influence on the control of drug-resistant virus. They also noted that the immune system can generate new CTL specificities even in patients with advanced disease, as the M184V HIV variant emerges only after drug treatment. However, Schmitt et al. could not determine whether these CTL in this 43-year-old patient were already present in the peripheral T cell repertoire or whether they were generated from lymphoid stem cells de novo. In the same study, Schmitt et al. reported that, although drug therapy was not able to suppress HIV-viraemia in this patient, plasma viral load remained stable at low levels and even declined over time gradually without change in antiviral therapy, suggesting that the HIV-1 specific immune response contributed to the control of HIV-1 in this patient. Schmitt et al. concluded that further studies are necessary to examine whether the induction of CTL against drug-escape variants can help to delay or even prevent the emergence of drug-resistant HIV-1 strains.
Although several approaches have been tried to overcome the problem of drug-resistant strains, most appear to simply delay the onset of resistance. A method addressing infection with drug-resistant strains is therefore a primary concern to health care providers.
In the present invention we demonstrate the use of a therapeutic HIV vaccine along with HIV antiviral therapy to develop CTLs specific for mutations that confer resistance to an antiviral drug in order to prevent such resistant mutants from occurring. These vaccines can also be optimized utilizing epitope enhancement.